Structure-function relationships in a glycosyltransferase, a phosphatase and an oxidoreductase

Abstract

Dissertation presented to obtain the Ph.D degree in BiochemistryEnzyme evolution is often constrained by aspects of catalysis. Mechanistically diverse enzymes evolved from a common ancestor still preserve those structural signatures essential to the core chemistry retained by all members of the superfamily. Indeed, these shared features allow superfamilies to be accurately classified, while derived features allow nested families and subfamilies to be identified in a hierarchical fashion. Accurate classification has helped elucidate mechanisms promoting functional diversification, for example catalytic promiscuity, and protein engineering by rational design. Nowadays, a holistic view of enzymes` regulatory mechanisms and catalytic proficiency is provided by the identification of conserved features of molecular architecture in combination with aspects of reaction dynamics. My work focused on the structural elucidation and analysis of three enzymes: a glycosyltransferase; a phosphatase and an oxidorreductase. “Snapshots” along the reaction coordinate of each enzyme were obtained by combining X-ray diffraction with “cryo-trapping” ligand-binding methods. These were used to characterize the molecular mechanisms involved in substrate recognition and binding. They were also used to distinguish between models proposed for the catalytic mechanisms of each enzyme, and provide insights into enzyme dynamics essential for catalysis and the stereo and regio-selective strategies at work.(...)Apoio financeiro da FCT e do POPH/FSE no âmbito do Quadro Comunitário de Apoio, Bolsa Nº SFRH/BD/23222/2005